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Identifier 000430224
Title Biochemical characterization of the CXXC- and PHD- fingers of the novel oncogene KDM2B.
Alternative Title Βιοχημικός χαρακτηρισμός των πρωτεϊνικών επικρατειών CXXC και PHD του ογκογονιδίου KDM2B
Author Δεικτάκης, Ελευθέριος
Thesis advisor Τσατσάνης, Χρήστος
Reviewer Καμπράνης, Σωτήριος
Καρδάσης, Δημήτριος
Abstract The fine line between physiological and pathological cell fate does not only depend on DNA sequence but also on a multifaceted matrix of regulatory elements derived from chemical modifications on the histone proteins or the DNA itself. Specific histone and DNA interacting factors that contain dedicated reader domains for specific modifications or combinations of them mediate the interpretation of this regulatory information. Deciphering what is written on chromatin with posttranslational modifications, elucidating their potential role in pathological cellular states, identifying the army of proteins that associate with them and understanding the way of function of those key player proteins, has become an exponentially growing field of research. Successful efforts on identifying readers and writers of the methyl mark on histone tails introduced an broaden spectrum of methyl transferases and demethylases that coordinate spatially and temporally, resulting in a dynamic epigenome that affects not only chromatin structure but physiological functions such as cellular development and proliferation while it is associated with several human diseases and cancer. Up until now, more than 20 proteins have been found being able to remove methyl groups from lysine residues on histone tails and they are classified into two super families. The amine oxidase superfamily includes demethylases that require FAD as co-factor and the oxygenase superfamily consists of proteins of which the demethylase activity, derived from their Jumonji C (JmjC) domain, is dependent on α-ketoglutarate and Fe2+. In human, the lysine specific demethylases are divided into seven families (KDM1-7), and most of them have been addressed as key epigenetic regulators. Some of them constitute vital components of more elaborate protein complexes (i.e. PRCs, NuRD, CoREST, MMLs) and participants in an abundance of physiological cellular processes (i.e transcription, DNA replication, DNA repair, etc.) in several signaling pathways (i.e Notch, TGF-β, FGF, NF-κB). Deregulation in the expression of these factors has been interconnected with numerous cases of cancer, leukemia and human tumorigenesis, suggesting a bivalent role as both oncogenic and tumor suppressor proteins. One such case is the lysine-specific demethylase 2B (KDM2B), a major PRC1-associated factor that targets H3K36me2, H3K4me3 and H3K79me2/3. Upon PRC2 recruitment, KDM2B participates in the repression of the senescence-associated expression genes. KDM2B functions by coupling several chromatin modifications, including histone H3K36me2 demethylation, with histone H3K27 trimethylation and histone H2AK119 monoubiquitination. Numerous studies revealed that KDM2B has a central role in occurrences of colon, prostate and pancreatic cancer and leukemogenesis. In addition, KDM2B functions as a master regulator of a set of microRNAs that target several members of the Polycomb complexes PRC1 and PRC2 and its deregulation has important effects on PRC gene expression in both normal and cancer cells. KDM2B has also been associated with inhibition of NF-κB/p65-dependent cellular apoptosis, by a mechanism where NF-κB upregulates KDM2B expression, resulting in the repression of c-FOS and the interception of apoptosis in human cancer cells. Moreover, KDM2B has been suggested to regulate genes of the glycolytic pathway and proteoglycan synthesis, as well as several other metabolic, antioxidant and pluripotency genes during morphogenesis and development. The protein structure of the KDM2B includes at the N-terminal the JmjC domain that responsible for the histone demethylation reaction and at the C-terminal several leucine-rich regions (LRRs) and an F-box domain that participate in protein-protein interactions. In addition, KDM2B contains two Zn2+ finger motifs, CxxC and PHD (Plant homeodomain), located at the center of the amino acid sequence. There is strong structural interdependence between these domains that prevents either of the two from being produced independently in a stable form. The KDM2B CxxC finger has been implicated in the DNA binding and the recognition of non-methylated CpG DNA sequences, however the structural determinants responsible for this interaction remain unclear. As far as the KDM2B PHD finger is concerned, it is considered the facilitator of KDM2B interactions with chromatin, however there have been several conflicting arguments about the substrate specificity of this domain, and its precise role is still elusive. In the current thesis, we set out to elucidate the role of the two Zn2+ finger domains of KDM2B, CxxC and PHD, in order to improve our understanding, on a molecular level, of their involvement in the KDM2B function that would enable us to develop a high-throughput domain-specific inhibitor-screening assay for KDM2B. Following this domain-wise approach, we cloned in a bacterial expression vector the CxxC-PHD coding sequence of the mouse KDM2B and the recombinant protein was submitted in a series of biochemical and biophysical assays. A series of side-directed mutageneses created several CxxC finger and PHD finger mutants that were used to identify the key residues involved in the interactions of these domains. Using electrophoresis mobility shift assays, we confirmed the Mg2+- independent binding of mKDM2B to non-methylated CpG-containing DNA sequences and we identified residues R585, K608 and K616 as key players in this interaction. The mKDM2B mutants that contained point mutations of those residues showed up to 24-fold reduced DNA binding capacity. The identification of the structural elements in the CxxC finger of KDM2B that participate in the DNA binding mechanism enabled us to correlate its function with its biological role in replicative senescence bypass in MEFs. Furthermore, we examined the role of this domain in the process of cellular migration of prostate cancer cells, by employing in vitro wound healing assays. Our results showed that the overexpression of the mKDM2B that carried the K616A mutation failed to induce the increased motility that was illustrated by the cells overexpressing the wild type KDM2B, suggesting that DNA recognition may be vital during metastasis and adhesion of cancer cells. With the view of our findings on the KDM2B CxxC finger, we aimed to develop a CxxC-targeting inhibitor screening by setting up a fluorescence-based assay that would provide a fast and easy way to assess the DNA recognition. The recombinant mKDM2B was fused with enhanced green fluorescent protein and a series of fluorescence resonance energy transfer experiments were designed. We managed to observe FRET between KDM2B-EGFP and DNA sequences that carried a Cy3 fluorophore, however, despite our best efforts, setting up a robust inhibitor-screening assay turned out to be unfeasible at that time due to several limitations set by the technical equipment. As far as the KDM2B PHD finger is concerned, we employed the technology of MODified® Histone Peptide Array that enabled us to examine 384 unique histone modification combinations as possible interactors. We confirmed the previously reported interaction of the KDM2B PHD domain with H3K4me3 (KD= 370 mM) and we revealed an unprecedented interaction with H4K20me3 (KD= 3 mM) and a weaker interaction with H2B tail spanning from residues 1 to 19. The ability of KDM2B PHD finger to recognize a modification mark such as H3K4me3 that is associated with euchromatin, and H4K20me3 that is a hallmark of silencing in some cases and of cancer in others, supports vividly the notion that this demethylase has pleiotropic functions that depend on the temporal and spatial intracellular context. In addition, the relatively high KD measured in the ITC experiments suggests that the overall binding of KDM2B with chromatin might be facilitated by additional interactions. Further investigation on the structural elements that participate in the histone recognition, illustrated that the phenylalanine residue (F654) that is placed in the expected active site of the PHD finger based on the in silico analysis, is not associated for this function, since the point-mutated protein successfully recognized H3K4me3 and H4K20me3, similarly to the wild type. Based on these results, we sought to investigated whether the substrate specificity of KDM2B PHD finger is similar to its sister protein, KDM2A. Comparative analysis of the MODified Histone Peptide Array results from both PHD fingers showed that KDM2A and KDM2B have different substrates, which is consistent with previous studies that have supported the idea of distinct and unrelated roles between these two demethylases. As a conclusion, the characterization of the molecular interactions of the CxxC and PHD fingers of KDM2B that is presented in this thesis provides a more explicit idea of their role and contribution to the function of this significant epigenetic factor and creates contemporary paths for further research.
Language English
Subject Lysine specific demethylase
Απομεθυλάση ιστόνων
Μοριακές αλληλεπιδράσεις
Issue date 2020-08-05
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work
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